Signaling mechanisms in tumor necrosis factor alpha-induced death of microvascular endothelial cells of the corpus luteum

Rueda, Pru, Lynch, Davis

Description

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F 2α (PGF 2α ) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF 2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways ( e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.

Sujets

• Apoptosis
• Corpus luteum
• Cytokine
• Endothélium
• Tumor necrosis factor
• Formal Approaches to Slavic Linguistics

Informations

• Publié par: biomed
• Publié le: 01 janvier 2003
• Nombre de lectures: 4
• Langue: English

Extrait

Reproductive Biology and
BioMed CentralEndocrinology
Open AccessResearch
Signaling mechanisms in tumor necrosis factor alpha-induced death
of microvascular endothelial cells of the corpus luteum
1 1 2 1James K Pru , Maureen P Lynch , John S Davis and Bo R Rueda*
1Address: Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston,
2Massachusetts 02114, USA and Olson Center for Women's Health, Department of Obstetrics and Gynecology, University of Nebraska Medical
Center, Omaha, Nebraska 68198; VA Medical Center, Omaha, Nebraska 68105, USA
Email: James K Pru - [e-mail]; Maureen P Lynch - [e-mail]; John S Davis - [e-mail];
Bo R Rueda* - [e-mail]
* Corresponding author
Published: 11 February 2003 Received: 27 January 2003
Accepted: 11 February 2003
Reproductive Biology and Endocrinology 2003, 1:17
This article is available from: http://www.RBEj.com/content/1/1/17
© 2003 Pru et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media
for any purpose, provided this notice is preserved along with the article's original URL.
apoptosiscorpus luteumcytokineendothelialtumor necrosis factorPGF2αFasL
Abstract
The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total
number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis
during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor
necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating
antibody (FasAb), and the luteolytic hormone prostaglandin F (PGF ) on CL-derived endothelial2α 2α
(CLENDO) cells. Neither sFasL, FasAb nor PGF had any effect on CLENDO cell viability. Utilizing2α
morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis
in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis
at early time points. Unlike many other studies described in non-reproductive cell types, TNFα
induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis.
TNFαinduced death is typically associated with acute activation of distinct intracellular signaling pathways
(e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs
(ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin
hydrolysis, can serve as an upstream activator of members of the MAPK family independently in
numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα,
treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis.
Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment
of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of
reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced
apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen
species, and intracellular signals that promote apoptosis.
may contribute to the high incidence of spontaneousBackground
Progesterone derived from the corpus luteum (CL) is re- abortions that occur early in mammalian pregnancies [3].
quired for the establishment and maintenance of a suita- In most species, the luteolytic cascade, starting with loss of
ble uterine environment during early pregnancy [1,2]. progesterone production and followed by tissue
regresInappropriate luteal function (i.e. luteal insufficiency) sion (i.e. apoptosis), is initiated by prostaglandin F2α
Page 1 of 11
(page number not for citation purposes)Reproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/17
(PGF ) in vivo [4]. However, this process can only be par- Interestingly it has been demonstrated that rapid produc-2α
tially recapitulated in vitro using primary cultures of ster- tion of ROS in endothelial cells exposed to TNFα is
medioidogenic cells. Gonadotropin-induced progesterone ated by ceramide [32]. Ceramide is a central figure in the
production is attenuated by PGF treatment, but this sphingomyelin pathway, which has also been linked to2α
prostanoid does not activate apoptosis of bovine ster- stress related signaling and apoptosis [33,34]. This report
oidogenic cells in vitro [8]. These findings helped develop provides evidence that cytokines regulate the fate of
the concept that additional factors, such as immune cells CLENDO cells. In the first set of experiments, we
identi[5–7] or cytokines [7,8], contribute to the luteolytic proc- fied those factors that initiate CLENDO cell death
ess. Indeed, tumor necrosis factor α (TNFα), interferon amongst a host of cytokines and lipid signaling molecules
gamma (IFNγ) and/or interleukin-1 beta (IL-1β) have all that mediate various aspects of luteal structural regression.
been shown to reduce gonadotrophin-induced progester- In a second series of experiments, we evaluated the
activaone synthesis in a number of species [8–12], and TNFα tion of several signal transduction pathways known to
coand IFNγ are known to increase PGF production by ster- ordinate apoptosis in other cell types. A working2α
oidogenic cells [13–15]. Steroidogenic cell apoptotic par- knowledge of the collective signaling network activated in
adigms are activated by cytokines generated by several cell CLENDO cells by specific death stimuli is essential to
types within the CL, including immune cells and endothe- complete our understanding of how apoptosis is
controllial cells [7,11,16]. The synergistic actions of IFNγ and TN- led within the CL.
Fα, for example, induce steroidogenic cell apoptosis
[13,17], as does soluble Fas ligand (FasL) [18–26]. Fur- Materials and Methods
thermore, several acute intracellular signaling events ei- Cell culture and treatments
ther required for, or associated with, the functional and Purified bovine CL-derived microvascular endothelial cell
structural aspects of luteal regression have been mapped populations were generated commercially by Cambrex
Biusing in vitro steroidogenic cell cultures [8,27,28]. osciences (BioWhittaker, Walkersville, MD) as described
in detail [35]. In the present study, CLENDO cells from
2The microvasculature of the CL is thought to be the first frozen aliquots (passage 3) were seeded (5,000 cells/cm )
structure to undergo degeneration via apoptosis during in 6- or 24-well plates or 10 cm culture dishes. The cells
luteolysis [2,8]. In contrast to information available on were cultured in EGM-2MV, as recommended by the
supsteroidogenic cells of the CL, a gap exists in our under- plier with progesterone (250 ng/ml) and 3% fetal bovine
standing of the factors and their associated signaling path- serum added. Once 90% confluency was reached, the
meways that initiate apoptosis in the vascular component of dium was changed and the cultures were maintained in
the CL. Elevated TNFα activity may play a role in the ap- serum- and growth factor-free conditions (EBM-2
medioptosis of CL-derived microvascular endothelial (CLEN- um) for 24 h. Medium was then removed once again and
DO) cells [29]. TNFα is known to initiate apoptosis in fresh serum- and growth factor-free medium was applied.
diverse cell types by activating its cognate receptor, TNF- Cells were allowed to equilibrate for a minimum of 3 h
RI, a cell surface receptor and member of the tumor necro- prior to treatment. Treatment concentrations were based
sis receptor super family (TNFRSF). TNF-RI is also a death upon previously published in vitro studies of mixed luteal
receptor based on the presence of a carboxy-terminal cells [13–15,17,29,36] and all experiments were
completfunctional domain known as the death domain. The ed a minimum of three times.
death domain serves as a binding domain for cytoplasmic
adapter proteins, which contain a death domain and a To identify putative luteolytic factors that initiate
CLENdeath effector domain (i.e. Fas associated death domain, DO cell apoptosis, cells were treated with TNFα (50 ng/
FADD)[30] ultimately activating members of the caspase ml; Upstate Biotechnology, Inc.; Lake Placid, NY), bovine
family resulting in apoptosis. Activation of TNFRI initiates IFNγ (200 IU/ml; generous gift from Dr. Dale Godson,
stress-activated pathways, which often are associated with Veterinary Infectious Disease Organization, University of
the onset of apoptosis in a cell specific fashion. Saskatchewan, Saskatoon, Saskatchewan, Canada), sFasL
(50 ng/ml; Oncogene, LaJolla, CA), mouse Fas activating
Of interest to these studies are the three subfamilies with- antibody (Jo2; 500 ng/ml; Pharmingen, San Diego, CA),
in the mitogen-activated protein kinase (MAPK) super- human Fas activating antibody (CH-11; 500 ng/ml;
Upfamily including the extracellular signal-related kinases state Biotechnology, Inc., Lake Placid, NY), PGF (1 µM;2α
(ERKs), p38 and c-jun N-terminal kinases (JNKs) [31]. Sigma Chemical Company; St. Louis, MO) or C2
ceraTNFα is one of many cytokines involved in luteal re